Lower eukaryotic host cells can be engineered to produce heterologous proteins. Further, lower eukaryotic host cells can be glyco-engineered to produce glycoproteins where the N- or O-linked glycosylation are modified from their native forms.
Engineered Pichia strains have been utilized as an alternative host system for producing recombinant glycoproteins with human-like glycosylation. However, the extensive genetic modifications have also caused fundamental changes in cell wall structures in many glyco-engineered yeast strains, predisposing some of these strains to cell lysis and reduced cell robustness during fermentation. Certain glyco-engineered strains have substantial reductions in cell viability as well as a marked increase in intracellular protease leakage into the fermentation broth, resulting in a reduction in both recombinant product yield and quality.
Current strategies for identifying robust glyco-engineered production strains rely heavily on screening a large number of clones using various platforms such as 96-deep-well plates, 5 ml mini-scale fermenters and 1 L-scale bioreactors to empirically identify clones that are compatible for large-scale (40 L and above) fermentation processes (Barnard et al. 2010). Despite the fact that high-throughput screening has been successfully used to identify several Pichia hosts capable of producing recombinant monoclonal antibodies with yields in excess of 1 g/L (Potgieter et al. 2009; Zhang et al. 2011), these large-scale screening approach is very resource-intensive and time-consuming, and often only identify clones with incremental increases in cell-robustness.
Therefore, lower eukaryotic host strains that have improved robustness and the ability to produce high quality proteins with human-like glycans would be of value and interest to the field. Here, we present engineered Pichia host strains having a deletion, truncation or nonsense mutation in a novel gene ATT1 (acquiring thermal tolerance) which under relevant bioprocess conditions exhibit improved viability, stability, and protein production. Surprisingly, engineered Pichia host strains over-expressing ATT1 or fragments thereof under relevant bioprocess conditions also exhibit improved viability, stability, and protein production. These strains are especially useful for heterologous gene expression.